Mutated TP53 in Circulating Tumor DNA as a Risk Level Biomarker in Head and Neck Squamous Cell Carcinoma Patients.

Circulating tumor DNA (ctDNA) has been suggested as a surrogate biomarker for early detection of cancer recurrence. We aimed to explore the utility of ctDNA as a noninvasive prognostic biomarker in newly diagnosed head and neck squamous cell carcinoma (HNSCC) patients. Seventy HNSCC specimens were analysed for the detection of TP53 genetic alterations utilizing next-generation sequencing (NGS). TP53 mutations were revealed in 55 (79%). Upon detection of a significant TP53 mutation, circulating cell-free DNA was scrutinized for the presence of the tumor-specific mutation. ctDNA was identified at a minimal allele frequency of 0.08% in 21 out of 30 processed plasma samples. Detectable ctDNA correlated with regional spread (N stage ≥ 1, p = 0.011) and poorer 5-year progression-free survival (20%, 95% CI 10.9 to 28.9, p = 0.034). The high-risk worst pattern of invasion (WPOI grade 4-5) and deep invasion were frequently found in patients whose ctDNA was detected (p = 0.087 and p = 0.072, respectively). Detecting mutated TP53 ctDNA was associated with poor progression-free survival and regional metastases, indicating its potential role as a prognostic biomarker. However, ctDNA detectability in early-stage disease and the mechanisms modulating its release into the bloodstream must be further elucidated.

Inflammatory mediators in breast cancer: coordinated expression of TNFα & IL-1ß with CCL2 & CCL5 and effects on epithelial-to-mesenchymal transition.

BACKGROUND: The inflammatory chemokines CCL2 (MCP-1) & CCL5 (RANTES) and the inflammatory cytokines TNFα & IL-1ß were shown to contribute to breast cancer development and metastasis. In this study, we wished to determine whether there are associations between these factors along stages of breast cancer progression, and to identify the possible implications of these factors to disease course. METHODS: The expression of CCL2, CCL5, TNFα and IL-1ß was determined by immunohistochemistry in patients diagnosed with: (1) Benign breast disorders (=healthy individuals); (2) Ductal Carcinoma In Situ (DCIS); (3) Invasive Ducal Carcinoma without relapse (IDC-no-relapse); (4) IDC-with-relapse. Based on the results obtained, breast tumor cells were stimulated by the inflammatory cytokines, and epithelial-to-mesenchymal transition (EMT) was determined by flow cytometry, confocal analyses and adhesion, migration and invasion experiments. RESULTS: CCL2, CCL5, TNFα and IL-1ß were expressed at very low incidence in normal breast epithelial cells, but their incidence was significantly elevated in tumor cells of the three groups of cancer patients. Significant associations were found between CCL2 & CCL5 and TNFα & IL-1ß in the tumor cells in DCIS and IDC-no-relapse patients. In the IDC-with-relapse group, the expression of CCL2 & CCL5 was accompanied by further elevated incidence of TNFα & IL-1ß expression. These results suggest progression-related roles for TNFα and IL-1ß in breast cancer, as indeed indicated by the following: (1) Tumors of the IDC-with-relapse group had significantly higher persistence of TNFα and IL-1ß compared to tumors of DCIS or IDC-no-relapse; (2) Continuous stimulation of the tumor cells by TNFα (and to some extent IL-1ß) has led to EMT in the tumor cells; (3) Combined analyses with relevant clinical parameters suggested that IL-1ß acts jointly with other pro-malignancy factors to promote disease relapse. CONCLUSIONS: Our findings suggest that the coordinated expression of CCL2 & CCL5 and TNFα & IL-1ß may be important for disease course, and that TNFα & IL-1ß may promote disease relapse. Further in vitro and in vivo studies are needed for determination of the joint powers of the four factors in breast cancer, as well as analyses of their combined targeting in breast cancer.

The Effect of Diathermy Power Settings on Oral Cavity Mucosal Contraction: A Rat Model.

OBJECTIVES/HYPOTHESIS: The effects of different electrocautery power settings on mucosal contraction and margin status in the oral cavity have not been well established. The aim of this study was to examine how different levels of electrocautery energy outputs affect oral mucosal tissue margins. STUDY DESIGN: Animal model. METHODS: A model of 23 adult rats was used (two specimens per rat). After anesthetizing the animals, a 6-mm biopsy punch marked the resection margin on the buccal mucosa (one per cheek). The specimens were excised by means of three energy levels, a cold knife, and monopolar diathermy that was set on either 20 W or 30 W cut modes. The specimens were evaluated for extent of contraction. RESULTS: A total of 45 samples were obtained and measured, including 15 specimens in the cold-knife group, 15 specimens in the 20 W group, and 15 specimens in the 30 W group. The median diameters of the specimens after resection were 4.5 mm for the cold-knife group (interquartile range [IQR] = 4.0-5.0), 3.5 mm for the 20 W group (IQR = 3.5-4.0), and 2.8 mm for the 30 W group (IQR = 2.5-3.0). Specimen contraction was 25.0%, 41.7%, and 53.3%, respectively. The difference in shrinkage between each pair was statistically significant: cold knife versus 20 W, P = .001; cold knife versus 30 W, P < .0001; and 20 W versus 30 W, P < .001. CONCLUSIONS: Diathermy power settings result in a significant difference of mucosal tissue contraction, with higher outputs resulting in a narrower mucosal margin. It is imperative that the surgical team take into consideration the diathermy settings during initial resection planning. Laryngoscope, 131:E1514-E1518, 2021.

Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient.

BACKGROUND: Neural stem cells are currently being investigated as potential therapies for neurodegenerative diseases, stroke, and trauma. However, concerns have been raised over the safety of this experimental therapeutic approach, including, for example, whether there is the potential for tumors to develop from transplanted stem cells. METHODS AND FINDINGS: A boy with ataxia telangiectasia (AT) was treated with intracerebellar and intrathecal injection of human fetal neural stem cells. Four years after the first treatment he was diagnosed with a multifocal brain tumor. The biopsied tumor was diagnosed as a glioneuronal neoplasm. We compared the tumor cells and the patient's peripheral blood cells by fluorescent in situ hybridization using X and Y chromosome probes, by PCR for the amelogenin gene X- and Y-specific alleles, by MassArray for the ATM patient specific mutation and for several SNPs, by PCR for polymorphic microsatellites, and by human leukocyte antigen (HLA) typing. Molecular and cytogenetic studies showed that the tumor was of nonhost origin suggesting it was derived from the transplanted neural stem cells. Microsatellite and HLA analysis demonstrated that the tumor is derived from at least two donors. CONCLUSIONS: This is the first report of a human brain tumor complicating neural stem cell therapy. The findings here suggest that neuronal stem/progenitor cells may be involved in gliomagenesis and provide the first example of a donor-derived brain tumor. Further work is urgently needed to assess the safety of these therapies.

NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis.

Cancer-Associated Fibroblasts (CAFs) were shown to orchestrate tumour-promoting inflammation in multiple malignancies, including breast cancer. However, the molecular pathways that govern the inflammatory role of CAFs are poorly characterised. In this study we found that fibroblasts sense damage-associated molecular patterns (DAMPs), and in response activate the NLRP3 inflammasome pathway, resulting in instigation of pro-inflammatory signalling and secretion of IL-1ß. This upregulation was evident in CAFs in mouse and in human breast carcinomas. Moreover, CAF-derived inflammasome signalling facilitated tumour growth and metastasis, which was attenuated when NLRP3 or IL-1ß were specifically ablated. Functionally, CAF-derived inflammasome promoted tumour progression and metastasis by modulating the tumour microenvironment towards an immune suppressive milieu and by upregulating the expression of adhesion molecules on endothelial cells. Our findings elucidate a mechanism by which CAFs promote breast cancer progression and metastasis, by linking the physiological tissue damage response of fibroblasts with tumour-promoting inflammation.

Concomitant expression of the chemokines RANTES and MCP-1 in human breast cancer: a basis for tumor-promoting interactions.

The chemokines RANTES (CCL5) and MCP-1 (CCL2) were suggested to contribute, independently, to breast malignancy. In the present study, we asked if the two chemokines are jointly expressed in clinical samples of breast cancer patients, and do they interact in breast tumor cells. We found that RANTES and MCP-1 were expressed by breast tumor cells in primary tumors of Ductal Carcinoma In Situ and of Invasive Ductal Carcinoma, but minimally in normal breast epithelial duct cells. The chemokines were also detected in metastases and pleural effusions. Novel findings showed that co-expression of RANTES and MCP-1 in the same tumor was associated with more advanced stages of disease, suggesting that breast tumors "benefit" from interactions between the two chemokines. Accordingly, MCP-1 significantly promoted the release of RANTES from endogenous pre-made vesicles, in an active process that depended on calcium from intracellular and extracellular sources, and on intracellular transport of RANTES towards exocytosis. Our findings show a chemokine-triggered release of stored pro-malignancy chemokine from breast tumor cells. These observations support a major tumor-promoting role for co-expression of the chemokines in breast malignancy, and agree with the significant association of joint RANTES and MCP-1 expression with advanced stages of breast cancer.

Ocular central nervous system lymphoma mimicking choroidal neovascularization.

Two patients evaluated for metamorphopsia were noted to have inferotemporal retinal pigment epithelium elevations formed by a yellowish lesion. Fluorescein angiography showed granular hyperfluorescence with late leakage, which was interpreted as an occult choroidal neovascularization. One patient underwent photodynamic therapy. In both patients, neither vitreous cells nor neurologic manifestations were evident on presentation. Subsequent neurological signs appeared that prompted performance of brain imaging, which confirmed a space-occupying lesion. In both patients, the tumor was proven on histopathologic examination of brain tissue to be central nervous system lymphoma. Awareness of other possible underlying pathologies is warranted in cases of atypical choroidal neovascularization.

Bone marrow-derived fibroblasts are a functionally distinct stromal cell population in breast cancer.

Cancer-associated fibroblasts (CAFs) are highly prominent in breast tumors, but their functional heterogeneity and origin are still largely unresolved. We report that bone marrow (BM)-derived mesenchymal stromal cells (MSCs) are recruited to primary breast tumors and to lung metastases and differentiate to a distinct subpopulation of CAFs. We show that BM-derived CAFs are functionally important for tumor growth and enhance angiogenesis via up-regulation of Clusterin. Using newly generated transgenic mice and adoptive BM transplantations, we demonstrate that BM-derived fibroblasts are a substantial source of CAFs in the tumor microenvironment. Unlike resident CAFs, BM-derived CAFs do not express PDGFRα, and their recruitment resulted in a decrease in the percentage of PDGFRα-expressing CAFs. Strikingly, decrease in PDGFRα in breast cancer patients was associated with worse prognosis, suggesting that BM-derived CAFs may have deleterious effects on survival. Therefore, PDGFRα expression distinguishes two functionally unique CAF populations in breast tumors and metastases and may have important implications for patient stratification and precision therapeutics.

The chemokine CCL5 as a potential prognostic factor predicting disease progression in stage II breast cancer patients.

PURPOSE: The aim of this study was to determine the prognostic value of the chemokine CCL5, considered as a promalignancy factor in breast cancer, in predicting breast cancer progression and to evaluate its ability to strengthen the prognostic significance of other biomarkers. EXPERIMENTAL DESIGN: The expression of CCL5, alone and in conjunction with estrogen receptor (ER)-alpha, ER-beta, progesterone receptor (PR), and HER-2/neu (ErbB2), was determined in breast tumor cells by immunohistochemistry. The study included 142 breast cancer patients, including individuals in whom disease has progressed. RESULTS: Using Cox proportional hazard models, univariate analysis suggested that, in stage I breast cancer patients, CCL5 was not a significant predictor of disease progression. In contrast, in stage II patients, the expression of CCL5 (CCL5(+)), the absence of ER-alpha (ER-alpha(-)), and the lack of PR expression (PR(-)) increased significantly the risk for disease progression (P = 0.0045, 0.0041, and 0.0107, respectively). The prognostic strength of CCL5, as well as of ER-alpha(-), improved by combining them together (CCL5(+)/ER-alpha(-): P = 0.0001), being highly evident in the stage IIA subgroup [CCL5(+)/ER-alpha(-) (P = 0.0003); ER-alpha(-) (P = 0.0315)]. In the stage II group as a whole, the combinations of CCL5(-)/ER-alpha(+) and CCL5(-)/PR(+) were highly correlated with an improved prognosis. Multivariate analysis indicated that, in stage II patients, ER-alpha and CCL5 were independent predictors of disease progression. CONCLUSIONS: CCL5 could be considered as a biomarker for disease progression in stage II breast cancer patients, with the CCL5(+)/ER-alpha(-) combination providing improved prediction of disease progression, primarily in the stage IIA subgroup.

A comprehensive algorithm for anterior skull base reconstruction after oncological resections.

OBJECTIVE: To present our method for anterior skull base reconstruction after oncological resections. METHODS: One hundred nine patients who had undergone 120 anterior skull base resections of tumors (52 malignant [43%], 68 benign [57%]) via the subcranial approach were studied. Limited dural defects were closed primarily or reconstructed using a temporalis fascia. Large anterior skull base defects were reconstructed by a double-layer fascia lata graft. A split calvarial bone graft, posterior frontal sinus wall, or three-dimensional titanium mesh were used when the tumor involved the frontal, nasal, or orbital bones. A temporalis muscle flap was used to cover the orbital socket for cases of eye globe exenteration, and a rectus abdominis free flap was used for subcranial-orbitomaxillary resection. Pericranial flap wrapping of the frontonaso-orbital segment was performed to prevent osteoradionecrosis if perioperative radiotherapy was planned. RESULTS: The incidence of cerebrospinal fluid (CSF) leak, intracranial infection, and tension pneumocephalus was 5%. Histopathological and immunohistochemical analysis of fascia lata grafts in reoperated patients (n = 7) revealed integration of vascularized fibrous tissue to the graft and local proliferation of a newly formed vascular layer embedding the fascial sheath. CONCLUSION: A double-layer fascial graft alone was adequate for preventing CSF leak, meningitis, tension pneumocephalus, and brain herniation. We describe a simple and effective method of anterior skull base reconstruction after resections of both malignant and benign tumors.

Efficacy of 780-nm laser phototherapy on peripheral nerve regeneration after neurotube reconstruction procedure (double-blind randomized study).

OBJECTIVE: This pilot double-blind randomized study evaluated the efficacy of 780-nm laser phototherapy on the acceleration of axonal growth and regeneration after peripheral nerve reconstruction by polyglycolic acid (PGA) neurotube. BACKGROUND DATA: The use of a guiding tube for the reconstruction of segmental loss of injured peripheral nerve has some advantages over the regular nerve grafting procedure. Experimental studies have shown that laser phototherapy is effective in influencing nerve regeneration. METHODS: The right sciatic nerve was transected, and a 0.5-cm nerve segment was removed in 20 rats. A neurotube was placed between the proximal and the distal parts of the nerve for reconnection of nerve defect. Ten of 20 rats received post-operative, transcutaneous, 200-mW, 780-nm laser irradiation for 14 consecutive days to the corresponding segments of the spinal cord (15 min) and to the reconstructed nerve (15 min). RESULTS: At 3 months after surgery, positive somato-sensory evoked responses were found in 70% of the irradiated rats (p = 0.015), compared to 30% of the non-irradiated rats. The Sciatic Functional Index in the irradiated group was higher than in the non-irradiated group (p < 0.05). Morphologically, the nerves were completely reconnected in both groups, but the laser-treated group showed an increased total number of myelinated axons. CONCLUSION: The results of this study suggest that postoperative 780-nm laser phototherapy enhances the regenerative process of the peripheral nerve after reconnection of the nerve defect using a PGA neurotube.

Multicentre validation of a microRNA-based assay for diagnosing indeterminate thyroid nodules utilising fine needle aspirate smears.

AIMS: The distinction between benign and malignant thyroid nodules has important therapeutic implications. Our objective was to develop an assay that could classify indeterminate thyroid nodules as benign or suspicious, using routinely prepared fine needle aspirate (FNA) cytology smears. METHODS: A training set of 375 FNA smears was used to develop the microRNA-based assay, which was validated using a blinded, multicentre, retrospective cohort of 201 smears. Final diagnosis of the validation samples was determined based on corresponding surgical specimens, reviewed by the contributing institute pathologist and two independent pathologists. Validation samples were from adult patients (≥18 years) with nodule size >0.5 cm, and a final diagnosis confirmed by at least one of the two blinded, independent pathologists. The developed assay, RosettaGX Reveal, differentiates benign from malignant thyroid nodules, using quantitative RT-PCR. RESULTS: Test performance on the 189 samples that passed quality control: negative predictive value: 91% (95% CI 84% to 96%); sensitivity: 85% (CI 74% to 93%); specificity: 72% (CI 63% to 79%). Performance for cases in which all three reviewing pathologists were in agreement regarding the final diagnosis (n=150): negative predictive value: 99% (CI 94% to 100%); sensitivity: 98% (CI 87% to 100%); specificity: 78% (CI 69% to 85%). CONCLUSIONS: A novel assay utilising microRNA expression in cytology smears was developed. The assay distinguishes benign from malignant thyroid nodules using a single FNA stained smear, and does not require fresh tissue or special collection and shipment conditions. This assay offers a valuable tool for the preoperative classification of thyroid samples with indeterminate cytology.

Fatal interstitial pneumonitis related to rituximab-containing regimen.

Rituximab, a chimeric anti-CD20 monoclonal antibody, is commonly being used to treat indolent and aggressive B-cell non-Hodgkin's lymphoma. Rituximab is considered a relatively safe drug, but recently, severe and fatal adverse effects related to this drug have been reported. In this regard, we report an 80-year-old patient with follicular grade 3 non-Hodgkin's lymphoma who developed a fatal interstitial pneumonitis related to treatment with a rituximab/CHOP (cyclophosphamide/doxorubicin/vincristine/prednisone) regimen. The pneumonitis was diagnosed on a routine midtreatment positron emission tomography/computed tomography scan when the patient was almost asymptomatic. Pulmonary deterioration occurred as the treatment with rituximab/CHOP was continued. In this article, we also review the literature on rituximab-associated pneumonitis, and we discuss the differential diagnosis with cyclophosphamide-induced lung injury.

Necrotizing Fasciitis of the Skull Base and Neck in a Patient with AIDS and Non-Hodgkin's Lymphoma.

Necrotizing fasciitis is a rapidly progressing, life-threatening soft tissue bacterial infection found more frequently in immunocompromised subjects and rarely in the head and neck. We report a rare case of a patient with acquired immunodeficiency syndrome (AIDS) and non-Hodgkin's lymphoma (NHL) who presented with a high fever and supraorbital cellulitis 1 week after undergoing chemotherapy. He received intravenous antibiotic therapy but soon developed dyspnea and trismus with rapid extension of the cellulitis to the face, ipsilateral infratemporal fossa (ITF), and bilateral neck. An awake tracheotomy was followed by surgical exploration and drainage and debridement of the supraorbital and ITF areas, parotid gland, and bilateral neck. He received intravenous antibiotic therapy and the surgical wound was regularly debrided for 10 days. Following a gradual recovery, the patient was discharged 2 weeks later. Early antibiotic therapy, wide surgical exploration, and a secured airway are the therapeutic mainstay for necrotizing fasciitis of the skull base and neck.

CXCR4 regulates migration and development of human acute myelogenous leukemia stem cells in transplanted NOD/SCID mice.

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 participate in the retention of normal hematopoietic stem cells within the bone marrow (BM) and their release into the circulation. Homing and engraftment of human stem cells in immunodeficient mice are dependent on cell surface CXCR4 expression and the production of BM SDF-1, which acts also as a survival factor for both human and murine stem cells. However, the role of SDF-1/CXCR4 interactions in the control of human acute myelogenous leukemia (AML) cell trafficking and disease progression is poorly understood. In this study, we report that although some AML cells do not express surface CXCR4, all AML cells tested express internal CXCR4 and SDF-1. Culture of AML cells with SDF-1 promoted their survival, whereas addition of neutralizing CXCR4 antibodies, SDF-1 antibodies, or AMD3100 significantly decreased it. Pretreatment of primary human AML cells with neutralizing CXCR4 antibodies blocked their homing into the BM and spleen of transplanted NOD/SCID/B2m(null) mice. Furthermore, weekly administrations of antihuman CXCR4 to mice previously engrafted with primary AML cells led to a dramatic decrease in the levels of human AML cells in the BM, blood, and spleen in a dose- and time-dependent manner. Interestingly, the same treatment did not affect significantly the levels of normal human progenitors engrafted into NOD/SCID mice. Taken together, our findings demonstrated the importance of the SDF-1/CXCR4 axis in the regulation of in vivo motility and development of human AML stem cells and identified CXCR4 neutralization as a potential treatment for AML.

Improving the rate of negative margins after surgery for oral cavity squamous cell carcinoma: A prospective randomized controlled study.

BACKGROUND: A positive margin is among the most significant factors that affects the outcome in head and neck squamous cell carcinoma (SCC). The purpose of this study was to compare the negative margin rates between 2 methods of intraoperative margin assessment in patients with oral cavity SCC. METHODS: A prospective, randomized controlled trial comparing 2 methods of intraoperative margin assessment: specimen-driven margins and patient-driven margins. RESULTS: The final analysis included 71 patients, 20 (29%) in the patient-driven margin arm. Frozen section analysis revealed positive/close surgical margins that led to an extension of the surgical resection in 22 of 51 patients (43%) in the specimen-driven margin arm, and 2 of 20 patients (10%) in the patient-driven margin arm (p = .01). After final pathological analysis, the wide negative margin rate was 84% in the specimen-driven margin arm, compared to 55% in the patient-driven margin arm (p = .02). Extension of the surgical resection prevented escalation of adjuvant treatment in 19 patients (38%) in the specimen-driven margin arm and 10% in the patient-driven margin arm. CONCLUSION: Specimen derived margin assessment led to significant improvement in the rate of negative margins. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1803-E1809, 2016.

Epidermal growth factor receptor gene amplification and expression in disseminated pediatric low-grade gliomas.

OBJECT: Pediatric low-grade gliomas (LGGs) are the largest group of central nervous system neoplasms in children. Although these tumors are generally benign, 5 to 10% of patients with pediatric LGGs present with leptomeningeal dissemination. The genetic and biological nature of these tumors is poorly understood. The authors looked for certain molecular abnormalities that may differentiate disseminated gliomas from the other pediatric LGGs. METHODS: Comparative genomic hybridization (CGH) was applied to 18 pediatric LGGs. Six cases featuring disseminated pediatric LGGs were compared with 12 control cases involving nondisseminated pediatric LGGs. Fluorescence in situ hybridization (FISH) analysis and immunohistochemical analysis were used to highlight further specific genetic targets. The CGH revealed multiple chromosomal abnormalities in five of six cases with disseminated gliomas and in six of 12 control cases. No correlation was found between the number of chromosomal abnormalities and dissemination status. Amplification of chromosome 7 was noted in four of six cases with disseminated gliomas as opposed to one of 12 control cases (p = 0.02). The FISH analysis revealed epidermal growth factor receptor (EGFR) amplification in one case negative to chromosome 7 amplification by CGH, raising the amplification cases to five of six (p = 0.0038). Immunohistochemical analysis for EGFR was positive in six of six cases and in two of 12 control cases (p = 0.0015). At the end of a mean follow-up period of 7.2 years, all patients with disseminated gliomas are alive with variable but slow disease progression. CONCLUSIONS: The high rate of EGFR gene amplification and protein expression in disseminated pediatric LGGs is intriguing and may have implications for our understanding of the role of EGFR in glioma genesis. Targeted therapies may be available for these children. Larger-scale studies are needed to establish further these findings.

Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway.

INTRODUCTION: Breast cancer progression is promoted by stromal cells that populate the tumors, including cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs). The activities of CAFs and MSCs in breast cancer are integrated within an intimate inflammatory tumor microenvironment (TME) that includes high levels of tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß). Here, we identified the impact of TNF-α and IL-1ß on the inflammatory phenotype of CAFs and MSCs by determining the expression of inflammatory chemokines that are well-characterized as pro-tumorigenic in breast cancer: CCL2 (MCP-1), CXCL8 (IL-8) and CCL5 (RANTES). METHODS: Chemokine expression was determined in breast cancer patient-derived CAFs by ELISA and in patient biopsies by immunohistochemistry. Chemokine levels were determined by ELISA in (1) human bone marrow-derived MSCs stimulated by tumor conditioned media (Tumor CM) of breast tumor cells (MDA-MB-231 and MCF-7) at the end of MSC-to-CAF-conversion process; (2) Tumor CM-derived CAFs, patient CAFs and MSCs stimulated by TNF-α (and IL-1ß). The roles of AP-1 and NF-κB in chemokine secretion were analyzed by Western blotting and by siRNAs to c-Jun and p65, respectively. Migration of monocytic cells was determined in modified Boyden chambers. RESULTS: TNF-α (and IL-1ß) induced the release of CCL2, CXCL8 and CCL5 by MSCs and CAFs generated by prolonged stimulation of MSCs with Tumor CM of MDA-MB-231 and MCF-7 cells. Patient-derived CAFs expressed CCL2 and CXCL8, and secreted CCL5 following TNF-α (and IL-1ß) stimulation. CCL2 was expressed in CAFs residing in proximity to breast tumor cells in biopsies of patients diagnosed with invasive ductal carcinoma. CCL2 release by TNF-α-stimulated MSCs was mediated by TNF-RI and TNF-RII, through the NF-κB but not via the AP-1 pathway. Exposure of MSCs to TNF-α led to potent CCL2-induced migration of monocytic cells, a process that may yield pro-cancerous myeloid infiltrates in breast tumors. CONCLUSIONS: Our novel results emphasize the important roles of inflammation-stroma interactions in breast cancer, and suggest that NF-κB may be a potential target for inhibition in tumor-adjacent stromal cells, enabling improved tumor control in inflammation-driven malignancies.

Localization of a specific germ cell marker, DAZL1, in testicular germ cell neoplasias.

DAZ-like 1( DAZL1) is a germ cell-specific protein expressed in both male and female gonads. The DAZL1 gene, which maps to chromosome 3 in humans, is an autosomal homologue to the Deleted in AZoospermia ( DAZ) gene(s) located on the Y chromosome. We studied the expression of DAZL1 by means of immunohistochemistry in order to determine its distribution among testicular germ cell neoplasias and among the intratubular lesions in their vicinity. Our results demonstrated that expression of DAZL1 protein was consistently observed in scattered cells in all intratubular germ cell neoplasias (IGCN) of the unclassified type, as well as in some of the intratubular seminomas. Foci of DAZL1 immunopositive cells were detected in pure seminomas, while single immunopositive cells were dispersed in the seminomatous component of mixed germ cell neoplasias. All the nonseminomatous components were negative for DAZL1 expression. These findings demonstrate an antigenic heterogeneity of seminoma cells. The localization of a specific germ cell protein, DAZL1, in the putative ontogenic progenitor, IGCN, and in their putative derivative, seminoma, provides further support for the hypothesis that IGCN is the precursor of germ cell neoplasias.

Cytogenetic analysis of three variants of clival chordoma.

Chordoma is an uncommon malignant neoplasm derived from remnants of the embryonal notochord. The tumor arises in the sacrococcygeal region in most cases. Cytogenetic information on clival chordomas is scarce due to the low incidence of these tumors. In this study, we applied the G-banding and spectral karyotyping techniques to compare the karyotypes of three variants of clival chordoma: conventional, chondroid, and dedifferentiated. We describe a normal karyotype of a chondroid chordoma and a complex karyotype of a conventional chordoma involving chromosomes 1, 2, 3, 5, 8, 9, 11, 15, 19, 20, and X. The cytogenetic analysis of the dedifferentiated chordoma showed a polyploid complex karyotype of 71-123 chromosomes with double minutes that originated from chromosome 17.